Uncover the latest breakthroughs in exosome research and their potential to transform regenerative medicine and immunotherapy. Don't miss this comprehensive exploration of the cutting-edge science behind extracellular vesicles and their impact on human health.

Extracellular vesicles (EVs) are a diverse group of membrane-bound particles released by cells into the extracellular environment. These vesicles play crucial roles in intercellular communication, transporting proteins, lipids, and nucleic acids between cells. This review aims to provide a detailed overview of EVs, covering their biogenesis, classification, cargo, functions, and potential therapeutic applications.

The Biology of Extracellular Vesicles (EVs): Biogenesis, Cargo, and Functions Explored

Extracellular Vesicles: Exosomes and Microvesicles

Extracellular vesicles (EVs) are membrane-bound particles released by cells into the extracellular environment. They play crucial roles in intercellular communication and have been implicated in various physiological and pathological processes. Two primary types of EVs are exosomes and microvesicles, which differ in their biogenesis, size, and release mechanisms.

Exosomes

Exosomes are small EVs, typically ranging from 30 to 150 nm in diameter. They originate from the endosomal pathway, specifically through the inward budding of the endosomal membrane, leading to the formation of multivesicular bodies (MVBs). When MVBs fuse with the plasma membrane, they release their intraluminal vesicles as exosomes into the extracellular space.

Biogenesis and Release

1. Endosomal Pathway: Exosomes are formed within the endosomal system. Early endosomes mature into late endosomes, which develop into MVBs containing intraluminal vesicles.
2. Intraluminal Vesicle Formation: The inward budding of the endosomal membrane forms intraluminal vesicles within MVBs. This process involves the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, although ESCRT-independent mechanisms exist.
3. MVB Fusion: MVBs can either fuse with lysosomes for degradation or with the plasma membrane to release exosomes into the extracellular space.

Endosomal Pathway for Exosome Production

The endosomal pathway is crucial for the biogenesis of exosomes, which are small extracellular vesicles involved in intercellular communication and various physiological processes. The production of exosomes involves several key steps and mechanisms, including the formation of multivesicular bodies (MVBs), the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, and alternative pathways such as the neutral sphingomyelinase (nSMase) pathway.

Key Steps in Exosome Production

1. Early Endosome Formation

The process begins with the formation of early endosomes through the internalization of cell surface proteins and lipids via endocytosis. Early endosomes serve as sorting hubs where cargo is directed either toward recycling back to the plasma membrane or toward degradation.

2. Maturation to Late Endosomes

Early endosomes mature into late endosomes, characterized by a more acidic environment and the presence of intraluminal vesicles (ILVs). This maturation involves the inward budding of the endosomal membrane, which encapsulates cytosolic components into ILVs.

3. Formation of Multivesicular Bodies (MVBs)

Late endosomes containing multiple ILVs are referred to as multivesicular bodies (MVBs). The formation of ILVs within MVBs is a critical step in exosome biogenesis and is regulated by the ESCRT machinery and other pathways.

Mechanisms Involved in ILV Formation

ESCRT Machinery

The ESCRT machinery is a complex of proteins that facilitates the sorting of ubiquitinated cargo and the inward budding of the endosomal membrane to form ILVs. The ESCRT complex is composed of several subunits (ESCRT-0, -I, -II, and -III) that sequentially assemble on the endosomal membrane to mediate cargo sorting and membrane scission.

Neutral Sphingomyelinase (nSMase) Pathway

An alternative pathway involves the enzyme neutral sphingomyelinase (nSMase), which generates ceramide, a lipid that promotes membrane curvature and budding. Inhibition of the nSMase pathway has been shown to impair exosome formation, indicating its significant role in ILV biogenesis and exosome production.

Fusion and Release of Exosomes

Once MVBs are formed, they can follow two main pathways:

1. Lysosomal Degradation: MVBs can fuse with lysosomes, leading to the degradation of their contents.
2. Exosome Release: Alternatively, MVBs can fuse with the plasma membrane, releasing their ILVs as exosomes into the extracellular space.

The decision between these pathways is influenced by various cellular signals and conditions. Efficient exosome release helps to modulate the endosomal pathway by alleviating potential "traffic jams" and unburdening the lysosomal system.

Regulation and Pathophysiological Implications

The integrity of the endosomal pathway is essential for proper exosome production. Dysfunctions in this pathway have been implicated in several neurodegenerative diseases, such as Alzheimer's disease (AD). For instance, the presence of the APOE4 allele, a genetic risk factor for AD, has been shown to reduce brain exosome production, potentially contributing to neuronal vulnerability and disease progression.

Intraluminal Vesicle Formation

Intraluminal vesicle (ILV) formation is a critical process in the biogenesis of multivesicular bodies (MVBs), which subsequently leads to the production of exosomes. This process involves the inward budding of the endosomal membrane, encapsulating various cargoes into vesicles within the lumen of MVBs. Several key mechanisms and pathways contribute to ILV formation, including the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, lipid-mediated pathways, and other regulatory proteins.

Key Mechanisms in ILV Formation

1. ESCRT Machinery

The ESCRT machinery is a series of protein complexes (ESCRT-0, -I, -II, and -III) that play a pivotal role in the sorting of cargo and the formation of ILVs. The process involves several steps:

• ESCRT-0: This complex recognizes and sequesters ubiquitinated cargo at the endosomal membrane.
• ESCRT-I and ESCRT-II: These complexes further concentrate the cargo and initiate membrane budding.
• ESCRT-III: This complex drives the final scission of the budding vesicle from the endosomal membrane, forming ILVs.

The ESCRT-III complex, in particular, is crucial for the abscission of ILVs into the lumen of MVBs. Proteins such as Vps68 have been shown to cooperate with ESCRT-III in this process, ensuring efficient vesicle formation.

2. Lipid-Mediated Pathways

Lipids play a significant role in membrane dynamics and ILV formation. One key lipid is ceramide, which is generated by the enzyme neutral sphingomyelinase (nSMase). Ceramide promotes membrane curvature and budding, facilitating the formation of ILVs independently of the ESCRT machinery. Additionally, phosphoinositolphosphates (PIPs), particularly phosphatidylinositol-4-phosphate (PI4P), have been implicated in regulating ILV formation by modulating the recruitment of proteins such as RAB10 to MVBs.

3. Other Regulatory Proteins

Several other proteins and pathways contribute to ILV formation:

• Clathrin: Clathrin and its associated proteins are involved in the timing and morphology of ILV formation, working in concert with the ESCRT machinery
• ER-Endosome Contact Sites: Membrane contact sites between the endoplasmic reticulum (ER) and endosomes play a role in regulating ILV formation. For example, the tyrosine phosphatase PTP1B, localized to the ER, interacts with endosomal targets to facilitate efficient sorting of cargo onto ILVs

Steps in ILV Formation

1. Cargo Recognition and Sequestration: Ubiquitinated cargo is recognized and sequestered by the ESCRT-0 complex at the endosomal membrane.
2. Membrane Budding Initiation: ESCRT-I and ESCRT-II complexes facilitate the budding of the endosomal membrane, concentrating the cargo into nascent ILVs.
3. Vesicle Scission: ESCRT-III drives the final scission of the budding vesicle, releasing the ILV into the lumen of the MVB.
4. Lipid Involvement: Lipids such as ceramide and PI4P modulate membrane curvature and protein recruitment, aiding in the formation and release of ILVs.

MVB Fusion in Exosome Formation

Multivesicular bodies (MVBs) play a crucial role in the biogenesis and release of exosomes. The process of MVB fusion with the plasma membrane is a key step that determines whether the intraluminal vesicles (ILVs) within MVBs are released as exosomes into the extracellular space or degraded in lysosomes. This fusion process involves several molecular mechanisms and regulatory proteins.

Key Steps in MVB Fusion

1. MVB Maturation

MVBs are formed from late endosomes that contain ILVs. These ILVs are generated through the inward budding of the endosomal membrane, capturing various cargoes such as proteins, lipids, and nucleic acids.

2. Transport to the Plasma Membrane

Mature MVBs are transported to the plasma membrane along the cytoskeleton. This transport is mediated by motor proteins such as kinesins and dyneins, which move MVBs along microtubules.

3. Docking and Tethering

Once MVBs reach the plasma membrane, they undergo a docking and tethering process. This step involves several proteins that help anchor the MVBs to the plasma membrane. Key players in this process include:

• Rab GTPases: These small GTP-binding proteins, such as Rab27a and Rab27b, regulate the docking of MVBs to the plasma membrane by interacting with effector proteins.
• SNARE Proteins: Soluble NSF Attachment Protein Receptors (SNAREs) are essential for the fusion of MVBs with the plasma membrane. Specific SNARE complexes facilitate the merging of the MVB membrane with the plasma membrane.

4. Membrane Fusion

The actual fusion of the MVB membrane with the plasma membrane is a highly regulated process that allows the release of ILVs as exosomes. This step involves:

• SNARE Complex Formation: The formation of a SNARE complex brings the membranes of the MVB and plasma membrane into proximity, facilitating membrane fusion.
• Calcium Influx: Calcium ions play a critical role in triggering membrane fusion. The influx of calcium can activate proteins such as synaptotagmins, which promote the fusion process.
• ESCRT Machinery: While the ESCRT machinery is primarily involved in ILV formation, components of the ESCRT complex can also participate in the final stages of MVB fusion with the plasma membrane.

Regulatory Proteins and Pathways

Several proteins and pathways regulate the fusion of MVBs with the plasma membrane:

• Rab GTPases: Rab27a and Rab27b are particularly important in the docking and fusion of MVBs. They interact with effectors like Munc13-4 and Slp4-a, which facilitate the tethering and fusion processes
• SNARE Proteins: The interaction between vesicle-associated SNAREs (v-SNAREs) on MVBs and target SNAREs (t-SNAREs) on the plasma membrane is crucial for membrane fusion
• Calcium Signaling: Calcium influx can trigger the fusion of MVBs with the plasma membrane by activating calcium-binding proteins such as synaptotagmins

Pathophysiological Implications

The fusion of MVBs with the plasma membrane and the subsequent release of exosomes have significant implications in various physiological and pathological processes:

• Cell-Cell Communication: Exosomes facilitate communication between cells by transferring bioactive molecules, influencing the behavior of recipient cells.
• Disease Progression: Dysregulation of exosome release can contribute to disease progression, including cancer, neurodegenerative diseases, and infectious diseases. For example, inhibiting exosome release has been shown to sensitize cancer cells to chemotherapy by preventing the export of drugs via exosomes
• Neurodegenerative Diseases: In neurodegenerative diseases, such as Parkinson's disease, the release of exosomes containing pathological proteins like α-synuclein can propagate disease pathology.

Functions and Significance

Exosomes carry a variety of bioactive molecules, including proteins, lipids, RNA, and DNA, which they can transfer to recipient cells, influencing various cellular processes. They are involved in:

- Cell-Cell Communication: Exosomes facilitate communication between cells by transferring their cargo, which can modulate the behavior of recipient cells.

- Disease Biomarkers: Due to their molecular content, exosomes are being explored as potential biomarkers for diseases such as cancer and neurodegenerative disorders[3].

- Therapeutic Potential: Exosomes are being investigated for their potential in drug delivery and regenerative medicine due to their ability to deliver therapeutic molecules to specific cells[2].

Exosomes carry a diverse array of bioactive molecules, including proteins, lipids, RNA, and DNA, which they can transfer to recipient cells, thereby influencing various physiological and pathological processes. The mechanisms of exosome-mediated communication involve the delivery of cargo such as enzymes, receptors, signaling molecules, mRNAs, microRNAs (miRNAs), other non-coding RNAs, bioactive lipids, and metabolites. These cargoes can modulate cellular pathways in recipient cells, regulating gene expression post-transcriptionally, and participating in signaling pathways.

Exosomes can be taken up by recipient cells through several mechanisms, including endocytosis (clathrin-mediated, caveolin-mediated, and macropinocytosis), direct fusion with the plasma membrane, and receptor-mediated uptake, which facilitates targeted delivery of their molecular content. In physiological processes, exosomes are involved in modulating immune responses by transferring antigens, cytokines, and other immune-modulatory molecules, presenting antigens to T cells, and influencing immune activation or suppression. In the nervous system, exosomes facilitate communication between neurons and glial cells, playing roles in synaptic plasticity, neuroprotection, and repair mechanisms. Additionally, exosomes derived from stem cells can promote tissue repair and regeneration by transferring growth factors and other regenerative molecules to damaged tissues. In pathological conditions, exosomes play significant roles in cancer progression by transferring oncogenic proteins and RNAs, modulating the tumor microenvironment, and facilitating metastasis. They can also contribute to drug resistance by exporting therapeutic agents out of cancer cells. In neurodegenerative diseases, exosomes can propagate pathology by transferring misfolded proteins such as amyloid-beta and alpha-synuclein between neurons. Furthermore, exosomes can modulate inflammatory responses by carrying pro-inflammatory or anti-inflammatory molecules, influencing the activation state of immune cells. Given their role in cell-cell communication, exosomes have significant potential in therapeutic and diagnostic applications.

They can be engineered to carry therapeutic agents, providing a natural and biocompatible delivery system that can target specific cells or tissues. The molecular content of exosomes reflects the state of their parent cells, making them valuable biomarkers for diagnosing diseases such as cancer, neurodegenerative disorders, and cardiovascular diseases. Exosomes derived from stem cells are being explored for their potential to promote tissue repair and regeneration, offering a promising approach to treating various degenerative diseases. In summary, exosomes are integral to intercellular communication, influencing a wide range of physiological and pathological processes through the transfer of bioactive molecules. Their ability to modulate immune responses, facilitate neuronal communication, and contribute to disease progression underscores their importance in both health and disease. The potential of exosomes in therapeutic and diagnostic applications continues to be a major area of research, promising new avenues for treatment and early detection of various conditions.

Microvesicles

Microvesicles, also known as ectosomes, are larger EVs ranging from 100 to 1,000 nm in diameter. They are formed by the outward budding and fission of the plasma membrane. This process is regulated by various signaling pathways and cytoskeletal rearrangements.

Biogenesis and Release

1. Plasma Membrane Budding: Microvesicles are generated through the outward budding of the plasma membrane. This involves the reorganization of the cytoskeleton and the redistribution of membrane lipids.
2. Regulatory Mechanisms: The formation and release of microvesicles are regulated by signaling pathways, including those involving calcium influx, which activates enzymes like calpain that facilitate membrane budding.
3. Fission: The final step involves the fission of the budded vesicle from the plasma membrane, releasing the microvesicle into the extracellular space.

Comparison of Exosomes and Microvesicles

Feature	Exosomes	Microvesicles
Size	30-150 nm	100-1,000 nm
Origin	Endosomal pathway	Plasma membrane
Biogenesis	Inward budding of the endosomal membrane	Outward budding of plasma membrane
Release Mechanism	Fusion of MVBs with plasma membrane	Direct budding and fission from the plasma membrane
Molecular Content	Proteins, lipids, RNA, DNA	Proteins, lipids, RNA, DNA
Functions	Cell-cell communication, biomarkers, therapeutic potential	Cell-cell communication, pathophysiological processes, diagnostic and therapeutic applications

Both exosomes and microvesicles are integral to cellular communication and hold promise for diagnostic and therapeutic applications. Understanding their distinct biogenesis and functional roles is crucial for harnessing their potential in clinical settings.

Apoptotic Bodies

Apoptotic bodies are larger EVs, ranging from 500 to 5,000 nm, produced during programmed cell death (apoptosis). They result from the blebbing of the plasma membrane and contain cellular organelles and nuclear fragments.

Classification of Extracellular Vesicles

EVs can be classified based on their size, density, and surface markers. The three main types are:

Exosomes: 30-150 nm, enriched in tetraspanins (CD9, CD63, CD81), Alix, and TSG101

Microvesicles: 100-1,000 nm, enriched in phosphatidylserine, integrins, and selectins

Apoptotic Bodies: 500-5,000 nm, containing cellular organelles and nuclear material

Cargo of Extracellular Vesicles

EVs carry a diverse array of bioactive molecules, including proteins, lipids, and nucleic acids. The specific cargo depends on the cell of origin and the physiological or pathological state of the cell.

Proteins

Extracellular vesicles (EVs) are lipid-bound vesicles secreted by cells into the extracellular space

The cargo of EVs consists of lipids, nucleic acids, and proteins, specifically proteins associated with the plasma membrane and cytosol. Quantitative proteomics has shown that EVs contain a variety of proteins, including cytoskeletal proteins, heat shock proteins, enzymes, and signaling molecules. Cytoskeletal proteins such as actin and tubulin are crucial for maintaining cell structure and facilitating intracellular transport. Heat shock proteins in EVs play a significant role in stress response and protein folding. Enzymes found in EVs are involved in various metabolic pathways and cellular processes. Signaling molecules within EVs, including tetraspanins and integrins, are essential for intercellular communication and signal transduction. The presence of these diverse proteins underscores the multifaceted roles of EVs in cellular physiology and pathology

Lipids

Extracellular vesicles (EVs) contain a lipid bilayer membrane that protects the encapsulated material, such as proteins, nucleic acids, lipids, and metabolites, from the extracellular environment

These vesicles are often enriched in cholesterol, sphingomyelin (SM), glycosphingolipids, and phosphatidylserine (PS). The formation of extracellular vesicles (EVs) is induced by the sphingolipid ceramide. Overexpression of CERT increases EV secretion while its inhibition reduces EV formation and the concentration of ceramides and sphingomyelins in EVs. Cholesterol accumulation in EVs contributes to their biogenesis, release, and uptake. Phosphatidylserine-positive extracellular vesicles interact especially with CD8+ T cells, providing antigen-specific adjuvant effects to activated CD8+ T cells in vivo

Nucleic Acids

Extracellular vesicles (EVs) carry various types of nucleic acids, including DNA, mRNA, and non-coding RNAs such as microRNAs and long non-coding RNAs. These nucleic acids play crucial roles in modulating gene expression in recipient cells. For instance, microRNAs within EVs can silence target mRNA transcripts through base-pair complementary binding, thereby regulating gene expression at the post-transcriptional level

Similarly, long non-coding RNAs, which are transcripts longer than 200 nucleotides, can influence cellular processes such as cell differentiation and development by modulating gene expression through various mechanisms depending on their localization within the cell. Moreover, mRNA molecules contained in EVs can be translated into proteins in recipient cells, directly impacting cellular functions and contributing to intercellular communication. The presence of these nucleic acids in EVs underscores their potential as non-invasive biomarkers for disease diagnosis and prognosis, as they reflect the molecular state of the originating cells and can induce specific biological responses in target cells

Functions of Extracellular Vesicles

EVs are involved in numerous physiological and pathological processes, including intercellular communication, immune modulation, and disease progression.

Intercellular Communication

EVs facilitate communication between cells by transferring their cargo, which can alter the behavior and function of recipient cells. This is particularly important in processes such as tissue repair, immune responses, and development.

Immune Modulation

EVs play a critical role in modulating the immune system. They can carry antigens, cytokines, and other immune-modulatory molecules, influencing the activation and suppression of immune responses.

Disease Progression

EVs are implicated in the progression of various diseases, including cancer, neurodegenerative disorders, and cardiovascular diseases. They can promote tumor growth, metastasis, and drug resistance by transferring oncogenic molecules and modulating the tumor microenvironment.

Role of Exosomes in Organ-Specific Functions

Brain

Exosomes play a pivotal role in the central nervous system (CNS) by mediating neuronal communication, synaptic plasticity, and neuroinflammation. They are involved in the transfer of neuroprotective and neurotoxic molecules, influencing the progression of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. For instance, exosomes derived from neurons can carry amyloid-beta peptides, contributing to the spread of amyloid plaques in Alzheimer's disease. Additionally, synaptic dysfunction, a hallmark of neurodegenerative diseases, has been linked to decreased levels of synaptic proteins in neuronal exosomes, as observed in patients with Alzheimer's disease and frontotemporal dementia. Hypoxic conditions in the CNS also affect exosome release, with hypoxic exosomes playing a role in injury and adaptation mechanisms, potentially exacerbating conditions like stroke and neurodegenerative diseases. Furthermore, plasma neuronal exosomes have been identified as biomarkers for cognitive impairment in Alzheimer's disease, reflecting the extent of synaptic damage and cognitive decline. The presence of specific microRNAs in neuronal exosomes, such as miR-373 and miR-204, has been associated with the regulation of inflammatory pathways in Alzheimer's disease, highlighting their potential as diagnostic markers and therapeutic targets.

Heart

Cardiac exosomes play a crucial role in heart development, repair, and disease by facilitating communication between cardiac cells and influencing processes such as angiogenesis, fibrosis, and inflammation. Exosomes derived from cardiac progenitor cells have been shown to prevent cardiomyocyte apoptosis through the delivery of miR-21, which targets PDCD4, thereby promoting cell survivalStudies have demonstrated that exosomes from pediatric cardiac progenitor cells significantly improve cardiac function, decrease fibrosis, and enhance angiogenesis following myocardial infarction, suggesting their reparative potential. Furthermore, pluripotent stem cell-derived exosomes have been recognized for their ability to promote cardiac repair and regeneration, offering promising translational applications for treating cardiovascular diseases. The paracrine effects of these exosomes, which include the transfer of cardioprotective microRNAs and proteins, are essential for modulating apoptosis, inflammation, fibrosis, and angiogenesis, thereby contributing to the overall regenerative capacity of the heart

Liver

Liver-derived exosomes play a multifaceted role in liver homeostasis, regeneration, and disease. Hepatocyte-derived exosomes are known to transfer lipids and proteins involved in metabolic processes, contributing to the maintenance of liver function and metabolic homeostasis

In the context of liver cancer, exosomes from liver cancer cells can promote tumor growth and metastasis by transferring oncogenic proteins and RNAs, thereby enhancing the malignant potential of cancer cells. Exosomes are also implicated in liver diseases such as hepatitis and cirrhosis, where they modulate immune responses and fibrogenesis. For instance, exosomes can carry viral components and immune-modulatory molecules that influence the progression of hepatitis. Additionally, in liver cirrhosis, exosomes can promote fibrogenesis by transferring fibrogenic signals to hepatic stellate cells, thereby contributing to the development of fibrosis. Furthermore, mesenchymal stem cell-derived exosomes have been shown to mitigate acute liver injury by upregulating protective genes such as Ets-1 and Heme Oxygenase-1, highlighting their potential therapeutic role in liver diseases

Kidney

Renal exosomes play a significant role in kidney function and pathology by participating in the regulation of renal cell communication, inflammation, and fibrosis. Exosomes derived from mesenchymal stem cells (MSCs) have shown potential in ameliorating acute kidney injury by delivering anti-inflammatory and regenerative molecules. For instance, exosomes from breast milk mesenchymal stem cells (Br-MSCs) have been found to mitigate adenine-induced nephropathy by modulating renal autophagy and fibrotic signaling pathways, as well as their epigenetic regulations, through the expression of antifibrotic miRNAs such as miR-181, miR-29b, and Let-7b

In kidney stone disease, macrophage-derived exosomes have been shown to enhance monocyte and T-cell migration, monocyte activation, and macrophage phagocytic activity, while also reducing T-cell activation and promoting the production of proinflammatory cytokines like IL-8. Furthermore, bone marrow mesenchymal stem cell (BMSC)-derived exosomes carrying microRNA-965 have been demonstrated to attenuate allogeneic renal transplant rejection by regulating the Janus kinase/signal transducers and activators of transcription 3 (JAK/STAT3) signaling pathway, thereby improving renal function and reducing histological changes in the kidney. Additionally, MSC-derived exosomes expressing C-C motif chemokine receptor-2 (CCR2) have been shown to suppress macrophage functions and alleviate ischemia/reperfusion-induced renal injury by acting as a decoy to suppress CCL2 activity

These findings underscore the therapeutic potential of exosomes in modulating renal inflammation, fibrosis, and overall kidney health.

Extracellular Vesicles in Disease: Exosome Roles in Cancer, Neurodegeneration, and More

Cancer

Exosomes play a multifaceted role in cancer by modulating tumor growth, metastasis, and the tumor microenvironment. Tumor-derived exosomes (TDEs) are actively produced and released by tumor cells and carry messages from tumor cells to healthy or abnormal cells, participating in tumor metastasis by transporting tumor-derived proteins and non-coding RNA to promote migration and angiogenesis. TDEs can transfer oncogenic proteins and RNAs to recipient cells, thereby promoting tumor proliferation and invasion, and they also facilitate immune evasion by modulating the activity of immune cells and decreasing MHC-I surface expression, as seen in esophageal cancer. Furthermore, exosomes derived from M2 type tumor-associated macrophages have been shown to promote drug resistance in non-small cell lung cancer through specific molecular pathways, highlighting their role in therapeutic resistance. The contents of TDEs reflect the phenotype of their cell of origin, making them valuable for disease diagnosis and evaluation, as they provide specific information about the genotype of a tumor and its response to treatment.

Cardiovascular Diseases

Exosomes play a significant role in the pathophysiology of cardiovascular diseases, including atherosclerosis, myocardial infarction, and heart failure. In the context of atherosclerosis, exosomes secreted from vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs), immune cells, and platelets facilitate intercellular communication, contributing to disease progression by transferring pro-inflammatory and pro-atherogenic molecules. For instance, platelet-derived exosomes (P-EXOs) are key mediators of inflammation and thrombosis, influencing the function of endothelial cells, leukocytes, and VSMCs, thereby exacerbating atherosclerosis. Conversely, exosomes derived from stem cells or cardioprotective cells hold therapeutic potential. For example, mesenchymal stem cell-derived exosomes containing lncRNA KLF3-AS1 can ameliorate pyroptosis of cardiomyocytes and myocardial infarction through the miR-138-5p/Sirt1 axis, promoting cardiac repair and reducing inflammation. Similarly, exosomes from cardiac fibroblast-induced pluripotent stem cells (CF-iPSCs) have shown promise in heart failure treatment by enhancing cardiac differentiation and function, partly through the modulation of miR22 levels, which are critical for cardiac hypertrophy and remodeling.

Neurodegenerative Diseases

In Alzheimer's disease (AD) and Parkinson's disease (PD), "the role of exosomes has been linked to the initiation and progression of these neurodegenerative diseases" by transporting neurotoxic proteins between cells, thereby spreading the pathology. Exosomes also play a significant role in modulating neuroinflammation, as they can carry and transfer inflammatory mediators that exacerbate or mitigate inflammatory responses in the brain. Furthermore, exosomes influence neuronal survival by transferring protective or harmful molecules, impacting cell viability and function. Therapeutic strategies targeting exosome biogenesis and uptake are being explored to mitigate the spread of neurotoxic proteins and promote neuroprotection. "The biogenesis of exosomes involves the inward budding of the endosomal membrane, leading to the formation of multivesicular bodies (MVBs)," and targeting this process could reduce the release of pathogenic exosomes.

Infectious Diseases

Exosomes are extracellular vesicles that play a pivotal role in the immune response to infectious diseases by transferring antigens and modulating immune cell activity

Pathogen-derived exosomes can influence host-pathogen interactions and contribute to disease progression by carrying pathogen-related molecules that mediate further infection and damage. For instance, exosomes from HIV-infected cells can transfer viral proteins and RNAs, which promote viral replication and immune evasion by targeting neighboring cells and activating pathways such as NF-κB, leading to cell proliferation and enhanced viral replication. This dual role of exosomes, in both spreading infection and activating immune responses, underscores their complexity and potential as therapeutic targets

Exosome-ECM Interactions

The extracellular matrix (ECM) is a non-cellular component present within all tissues and organs, providing essential physical scaffolding for cellular constituents and initiating crucial biochemical and biomechanical cues required for tissue morphogenesis, differentiation, and homeostasis

The ECM is composed of a complex network of proteins, polysaccharides, and water that comprise the acellular stromal microenvironment in all tissues and organs. Exosomes, a subtype of extracellular vesicles (EVs), interact with the ECM by carrying various regulatory proteins, nucleic acids, and lipids, which influence ECM remodeling and tissue repair. These interactions are critical for maintaining tissue homeostasis and play a significant role in disease processes, including cancer progression, by modulating ECM composition and structure. The dynamic interplay between exosomes and the ECM involves the secretion and uptake of exosomes, which can control ECM remodeling and affect cellular signaling pathways, thereby influencing cell migration, proliferation, and differentiation

ECM Remodeling

Exosomes can carry enzymes such as matrix metalloproteinases (MMPs) that degrade ECM components, facilitating tissue remodeling and repair. "Odontoblast media were also assayed for respective type III procollagen propeptide (PIIINP)"

They also deliver ECM proteins and regulatory molecules that influence ECM synthesis and organization. "The analysis of molecular mechanisms by which the ischemic myocardium initiates repair and remodeling indicates that secreted soluble factors are key players in communication to local and distant tissues". For instance, exosomes from M2c macrophages have been shown to promote ECM anabolism in intervertebral disc degeneration by delivering miR-124 and modulating the expression of ECM proteins. "To elucidate the underlying molecular mechanism, we performed 4D label‐free proteomics to screen dysregulated proteins in NPs treated with M2c‐Exoss"

Fibrosis

In fibrotic diseases, exosomes contribute to the deposition and remodeling of ECM by transferring pro-fibrotic molecules such as transforming growth factor-beta (TGF-β) and fibronectin. Exosomes are nanoscale vesicles capable of transporting a variety of molecules in the body and mediating intercellular communication

They can influence the activity of fibroblasts and myofibroblasts, promoting ECM synthesis and fibrosis. Exosomes from fibrotic tissues can also transfer fibrotic signals to neighboring cells, exacerbating the fibrotic response. The extensive proliferation, differentiation, and ECM deposition of myofibroblasts are triggered by a variety of cytokines and growth factors, such as TGF-β1, derived from adjacent cells or from fibroblasts themselves. This local ECM remodeling is associated with increased proliferation and motility of cells, contributing to the development of fibrosis. Exosomes derived from several cells, such as mesenchymal stem cells, have demonstrated potential as fibrotic agents by promoting fibroblast proliferation and collagen deposition. The pathologic matrix-producing cells are activated fibroblasts, called myofibroblasts, in different organ and tissue fibrosis. Exosomes can regulate the secretion of ECM by influencing the expression of matrix metalloproteinases (MMPs) and other factors

Cancer Metastasis

Exosomes play a pivotal role in cancer metastasis by modulating the extracellular matrix (ECM) and creating a pre-metastatic niche. Tumor-derived exosomes (TDEs) can transfer molecules that promote ECM degradation, angiogenesis, and immune suppression, facilitating tumor cell invasion and colonization of distant organs. Exosomes released by cancer cells precondition tissue environments for local spreading and distant metastasis by delivering inflammatory and other factors

These vesicles carry important molecules that affect ECM degradation and composition, aiding in proteolytic degradation of the ECM. TDEs are capable of modulating the tumor microenvironment and ECM by stimulating extracellular receptor signaling and disrupting cell adhesion formation. Exosomes from breast cancer promote the adhesion of cells to extracellular matrix proteins. Exosomal integrins participate in the initiation of cancer cell colonization and the formation of a pre-metastatic niche. Tumor-derived exosomes containing miR-21 or let-7a (under hypoxic stress) increase M2 polarization of macrophages, which may stimulate tumor-associated angiogenesis and lymphangiogenesis. Exosomes derived from cancer cells can also induce the differentiation of many types of tumor microenvironment cells to cancer-associated fibroblasts (CAFs), which are dominant in the tumor microenvironment and play a crucial role in ECM remodeling and TME reprogramming

Exosomes as Biomarkers and Therapeutics: The Potential of Extracellular Vesicles in Medicine

Drug Delivery

Exosomes are nanosized vesicles secreted by cells, attracting increasing interest in biomedical research due to their unique properties, including biocompatibility, cargo loading capacity, and deep tissue penetration

. They serve as natural signaling agents in intercellular communication, and their inherent ability to carry proteins, lipids, and nucleic acids endows them with remarkable therapeutic potential. Exosomes have been used as nano-carriers for small molecules and macromolecules (siRNA and pDNA) in cancer therapy in pre-clinical studies, as well as biomarkers for cancer diagnosis and prognosis. They can escape immune recognition and metabolic destruction, and they lack undesired accumulation before reaching their intended targets. Surface-modified exosomes are a potentially effective strategy, increasing circulation time and producing specific drug target vehicles. Exosomes loaded with anti-cancer drugs have shown promise in enhancing drug delivery and reducing off-target effects in cancer therapy. Despite their advantages, there are several challenges in optimizing cargo loading efficiency and structural stability and in defining exosome origins. Future research should include the development of large-scale, quality-controllable production methods, the refinement of drug loading strategies, and extensive in vivo studies and clinical trials

Regenerative Medicine

In regenerative medicine, exosomes from stem cells and progenitor cells are being investigated for their potential to promote tissue repair and regeneration. Exosomes play an important role in organism homeostasis and disease development, working as vehicles for the transfer of signaling and regulatory molecules between cells[1]. They can deliver regenerative molecules that enhance cell proliferation, differentiation, and survival, making them an attractive new tool for regenerative medicine[1]. Exosome-based therapies are being developed for conditions such as myocardial infarction, stroke, and chronic wounds, showing promising results in preclinical studies[1]. Mesenchymal stem cell-derived exosomes have demonstrated significant therapeutic potential in various diseases, including COVID-19, alopecia, skin aging, and osteoarthritis[1]. The regenerative capabilities of these exosomes are attributed to their cargo content, which reflects the physiological state of the parent cells[1].

Immunotherapy

Exosomes are being utilized in immunotherapy to modulate immune responses and enhance the efficacy of vaccines. They can carry antigens and immune modulatory molecules that activate immune cells and promote anti-tumor immunity. Exosome-based vaccines are being explored for cancer and infectious diseases, aiming to elicit robust and specific immune responses. Exosomes, as phospholipid extracellular vesicles secreted by various cells, contain non-coding RNA (ncRNA), mRNA, DNA fragments, lipids, and proteins, which are essential for intercellular communication

Tumor-derived exosomes (TDEs) express complexes of MHC class I/II epitopes and costimulatory molecules, playing a crucial role in the tumor immune microenvironment (TIME). Exosomes purified from macrophages treated with Mycobacterium tuberculosis culture filtrate proteins (CFP) were found to induce antigen-specific IFN-γ and IL-2-expressing CD4+ and CD8+ T cells, suggesting their potential as a novel cell-free vaccine against tuberculosis. Exosome-based vaccines derived from dendritic cells have shown promising results in inducing strong anti-tumor immune responses and are simpler and more cost-effective compared to traditional dendritic cell vaccines. Furthermore, macrophages releasing antigen-carrying exosomes enhance dendritic cell-mediated CD4+ T cell responses, illustrating a new crosstalk mechanism between macrophages and dendritic cells to regulate adaptive immunity

Challenges and Future Directions

Despite the promising potential of EVs, several challenges need to be addressed to fully harness their therapeutic applications. Standardization and characterization of extracellular vesicles (EVs) remain challenging due to the lack of consensus on isolation and quantification methods, which hinders reproducibility and clinical application

Developing robust protocols, such as those involving size exclusion chromatography and tunable resistive pulse sensing, is essential to ensure the efficient recovery of physiologically intact EVs for downstream analyses. Understanding the mechanisms of EV uptake by recipient cells is crucial, as EVs can enter cells through various endocytic pathways, including clathrin-dependent and independent routes, which influence their therapeutic potential. Further research into these mechanisms will enable the development of targeted EV-based therapies by elucidating how EVs alter recipient cell phenotypes. Scaling up the production of EVs while maintaining their quality and functionality is a significant challenge, necessitating the development of scalable and cost-effective methods such as bioreactor systems and physical or chemical stimulation to increase EV yield without compromising their therapeutic properties

References

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